0022-3565/02/3011-103–110$7.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
JPET 301:103–110, 2002
Vol. 301, No. 1
4525/969728
Printed in U.S.A.
Inhibitors of ATP-Binding Cassette Transporters Suppress
Interleukin-12 p40 Production and Major Histocompatibility
Complex II Up-Regulation in Macrophages
GYÖRGY HASKÓ, EDWIN A. DEITCH, ZOLTÁN H. NÉMETH, DAVID G. KUHEL, and CSABA SZABÓ
Department of Surgery, University of Medicine and Dentistry-New Jersey Medical School, Newark, New Jersey (G.H., E.A.D., Z.H.N.); and
Inotek Corporation, Beverly, Massachusetts (D.G.K., C.S.)
Received September 7, 2001; accepted December 11, 2001
This article is available online at http://jpet.aspetjournals.org
the suppressive effect of glibenclamide on IL-12 p40 production. On the other hand, both the MDR inhibitor verapamil and
CFTR blocker 2,2⬘-iminodibenzoic acid failed to suppress the
production of IL-12 p40. Furthermore, selective inhibitors and
activators of SURs were without effect. In agreement with the
pharmacological data, macrophages expressed mRNA for
ABC1, but not SURs or CFTR. Intracellular levels of IL-12 p40
were decreased by glibenclamide, suggesting that glibenclamide does not affect IL-12 p40 secretion. The effect of
glibenclamide did not involve an interference with the activation
of the p38 and p42/44 mitogen-activated protein kinases or
c-Jun kinase. Glibenclamide also suppressed IFN-␥-induced
up-regulation of major histocompatibility complex II. Taken together, our results indicate that ABC proteins regulate LPS
and/or IFN-␥-induced macrophage activation.
ATP-binding cassette (ABC) transporters are a large family of proteins that mediate the transport of a wide range of
substances across biological membranes (Higgins, 1995).
ABC proteins are defined by the presence of the ABC unit,
which contains two conserved peptide motifs (Walker A and
Walker B) that are able to bind ATP (Klein et al., 1999). As
membrane transporters, the ABC proteins also contain membrane-embedded transmembrane domains. The minimal
structural requirement for an active ABC protein is to have
two transmembrane domains and two ABC units (Klein et
al., 1999). More than 100 ABC proteins have now been cloned
in a variety of species, including bacteria and plants, as well
as mammals (Higgins, 1995). The best characterized ABC
proteins are the sulfonylurea receptors (SURs) 1 and 2, cystic
fibrosis conductance regulator (CFTR), multidrug resistance
protein (MDR), and Tangier disease protein ABC1. In addition to their structural similarity, SURs, CFTR, MDR, and
ABC1 are also similar in that their activity is selectively
inhibited by the sulfonylurea drug glibenclamide.
Recent data indicate that various members of the ABC
protein family are present in immune cells. For example,
MDR, a plasma-membrane glycoprotein that confers multidrug resistance on tumor cells, is expressed in cells of the
immune system, including macrophages and lymphocytes
(Hughes et al., 1983). CFTR has been found in both human
macrophages and neutrophils (Yoshimura et al., 1993). Another member of the ABC family, the TAP-1/TAP2 peptide
transporter, is involved in antigen presentation (Marusina
and Monaco, 1996). A novel member of ABC proteins, ABC1,
has recently been shown to be expressed by cells of the
monocyte/macrophage lineage (Luciani and Chimini, 1996;
Langmann et al., 1999). ABC1 is required for engulfment of
cells undergoing apoptosis by macrophages and it is involved
in the translocation of phospholipids and cholesterol to
ABBREVIATIONS: ABC, ATP-binding cassette; SUR, sulfonylurea receptor; CFTR, cystic fibrosis conductance regulator; MDR, multidrug
resistance protein; IL, interleukin; DIDS, diisothiocyanostilbene-2,2⬘-disulfonic acid; BSP, sulfobromophthalein; IFN, interferon; LPS, lipopolysaccharide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DPC, 2,2⬘-iminodibenzoic acid; DMSO, dimethyl sulfoxide; PBS,
phosphate-buffered saline; TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; RT-PCR, reverse transcription-polymerase
chain reaction; PCR, polymerase chain reaction; bp, base pair; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal protein kinase;
I␬B, inhibitory factor ␬B; Ab, antibody; MHC, major histocompatibility complex; KATP, ATP-gated potassium.
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ABSTRACT
ATP-binding cassette (ABC) transporters are a large family of
proteins whose role is to translocate various substances across
biological membranes. They include the Tangier disease protein ABC1, sulfonylurea receptors (SUR), multidrug resistance
protein (MDR), and cystic fibrosis transmembrane regulator
(CFTR). In the current study, we investigated the involvement of
ABC transporters in the regulation of lipopolysaccharide (LPS)
and/or interferon (IFN)-␥-induced interleukin (IL)-12 p40 and
tumor necrosis factor (TNF)-␣ production, nitric oxide formation, as well as major histocompatibility complex II up-regulation in macrophages. The general ABC transporter inhibitor
glibenclamide suppressed both IL-12 p40 and nitric oxide production. However, glibenclamide failed to affect the production
of TNF-␣. The selective ABC1 inhibitors 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid and sulfobromophthalein mimicked
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Haskó et al.
Materials and Methods
Mice. Male BALB/c mice (8 weeks) were purchased from Charles
River Laboratories, Inc. (Wilmington, MA).
Reagents and Drugs. Lipopolysaccharide (LPS; Escherichia coli
serotype 055:B5), DIDS, BSP, agarose, thioglycollate medium, and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
were purchased from Sigma-Aldrich (St. Louis, MO). Glibenclamide
(N-p-[2-(5-chloro-2-methoxybenzamido)ethyl]benzene-sulfonyl-N⬘cyclohexylurea), glipizide, tolbutamide, pinacidil, diazoxide, and minoxidil were obtained from Sigma/RBI (Natick, MA). 2,2⬘-Iminodibenzoic acid (DPC) was purchased from Aldrich Chemical (Milwaukee, WI). Glibenclamide, pinacidil, diazoxide, glipizide, minoxidil,
DIDS, and BSP were dissolved in DMSO, with a 0.5% final DMSO
concentration in the medium. RPMI-1640, F-12K medium, fetal bovine serum, and penicillin-streptomycin were obtained from Invitrogen (Carlsbad, CA).
Cell Lines. The J774, RAW 264, and NIT-1 cell lines were obtained from American Type Culture Collection (Manassas, VA). The
mouse macrophage cell lines J774 and RAW 264 were grown in
RPMI-1640 or Dulbecco’s modified Eagle’s medium supplemented
with 10% fetal bovine serum, 100 U/ml penicillin, and 100 ␮g/ml
streptomycin in a humidified atmosphere of 95% air and 5% CO2.
The mouse insulinoma cell line NIT-1 was cultured in F-12K medium
supplemented with 10% heat inactivated fetal bovine serum and 100
U/ml penicillin, and 100 ␮g/ml streptomycin.
Preparation of Peritoneal Macrophages. Mice were injected
intraperitoneally with 2 ml of 2% thioglycollate and peritoneal cells
were harvested 3 to 4 days later. The cells were plated on 96-well plastic
plates at 1 million cells/ml and incubated in RPMI-1640 for 2 h at 37°C
in a humidified 5% CO2 incubator. Nonadherent cells were removed by
rinsing the plates three times with 5% dextrose in PBS.
Treatment of J774 Cells and Peritoneal Macrophages. Cells in
96-well plates were treated with various concentrations of ABC inhibitors 30 min before the addition of 10 ␮g/ml LPS and 100 U/ml IFN-␥ or
10 ␮g/ml LPS. Twenty-four hours after stimulation with LPS or LPS/
IFN-␥, supernatants were taken for IL-12 p40, tumor necrosis factor
(TNF)-␣, and nitric oxide determination. For the determination of intracellular IL-12 p40 and TNF-␣, J774 macrophages in 12-well plates
were pretreated with glibenclamide followed by LPS/IFN-␥ stimulation
30 min later. After an additional 24-h incubation, the supernatants
were removed and the cells were lysed using 200 ␮l of modified radioimmunoprecipitation buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM
EDTA, 0.25% Na-deoxycholate, 1% Nonidet P-40, 1 ␮g/ml pepstatin, 1
␮g/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4).
IL-12 p40 and TNF-␣ levels in cell supernatants or cell lysates were
determined by ELISA as described below.
Cytokine Assays. Cytokine concentrations were determined by
ELISA kits that are specific against murine IL-12 p40 or TNF-␣.
Levels of IL-12 p40 and TNF-␣ were measured using ELISA kits
purchased from Genzyme (Boston, MA). Plates were read at 450 nm
by a Spectramax 250 microplate reader from Molecular Devices
(Sunnyvale, CA). The detection limit was 10 pg/ml. Assays were
performed according to the manufacturer’s instructions.
RNA Isolation and RT-PCR. Total RNA was isolated from
mouse heart, spleen, and kidney, as well as from the J774, RAW264,
and NIT-1 cells by using TRIzol Reagent (Invitrogen). Reverse transcription of the RNA was performed using 50 U/␮l MuLV reverse
transcriptase from PerkinElmer (Foster City, CA). RNA (5 ␮g) was
transcribed in a 20-␮l reaction containing 10.7 ml of RNA, 2 ␮l of
10⫻ PCR buffer, 2 ␮l of 10 mM dNTP mix, 2 ␮l of 25 mM MgCl2, 2
␮l of 100 mM dithiothreitol, 0.5 ␮l of RNase inhibitor (20 U/␮l;
PerkinElmer), 0.5 ␮l of 50 ␮M oligo d(T)16 (with the exception of
SUR2 and CFTR, where the antisense primer for PCR amplification
was used), and 0.3 ␮l of reverse transcriptase. The reaction mix was
incubated at 42°C for 15 min for reverse transcription. Thereafter,
the reverse transcriptase was inactivated at 99°C for 5 min. RTgenerated DNA (1–5 ␮l) was amplified using Expand high-fidelity
PCR system (Roche Molecular Biochemicals, Indianapolis, IN). The
reaction buffer (25 ␮l) contained 1 to 5 ␮l cDNA, water, 2.5 ␮l of PCR
buffer, 1.5 ␮l of 25 mM MgCl2, 1 ␮l of 10 mM dNTP mix, 0.5 ␮l of 10
␮M oligonucleotide primer (each), and 0.2 ␮l of enzyme. cDNA was
amplified using the following primers and conditions: SUR1, 5⬘ATTAACCTGAGAGGGGCGAT-3⬘ (sense) and 5⬘-GAGGTGTAGACAGCGAAGGC-3⬘ (antisense), an initial denaturation at 94°C ⫻ 5
min, 35 cycles of 94°C ⫻ 30 s, 58°C ⫻ 45 s, 72°C ⫻ 45 s, and a final
dwell at 72°C ⫻ 7 min; SUR2A/B, 5⬘-TGCGACATTTGTGACACATG-3⬘ (sense) and 5⬘-CGTAAGCCACAGAATACCTGC-3⬘ (antisense), an initial denaturation at 94°C ⫻ 5 min, 35 cycles of 94°C ⫻
30 s, 58°C ⫻ 45 s, 72°C ⫻ 45 s, and a final dwell at 72°C ⫻ 7 min;
Kir6.1, 5⬘-GCAAACCCGAGTCTTCTAGG-3⬘ (sense) and 5⬘-GCAGACGTGAATGACCTGAC-3⬘ (antisense), an initial denaturation at
94°C ⫻ 5 min, 35 cycles of 94°C ⫻ 30 s, 56°C ⫻ 30 s, 72°C ⫻ 45 s, and
a final dwell at 72°C ⫻ 7 min; Kir6.2, 5⬘-CTGGCCATCCTCATTCTC-3⬘ (sense) and 5⬘-GATGCCCGTGGTTTCTAC-3⬘ (antisense),
an initial denaturation at 94°C ⫻ 5 min, 38 cycles of 94°C ⫻ 30 s,
57°C ⫻ 30 s, 72°C ⫻ 45 s, and a final dwell at 72°C ⫻ 7 min; ABC1,
5⬘-GGAGTCTAGTCCTCTTTCTC-3⬘ (sense) and 5⬘-CCATGAATCGAGATATCGTC-3⬘ (antisense), an initial denaturation at 94°C ⫻ 5
min, 38 cycles of 94°C ⫻ 30 s, 58°C ⫻ 45 s, 72°C ⫻ 45 s, and a final
dwell at 72°C ⫻ 7 min; CFTR (Marvao et al., 1998), 5⬘-CAGTCATCTCTGCCTTGTGGGA-3⬘ (sense) and 5⬘-CGAACTGAAGCTCGGACGTAGACT-3⬘ (antisense), an initial denaturation at
94°C ⫻ 5 min, 35 cycles of 94°C ⫻ 30 s, 60°C ⫻ 30 s, 72°C ⫻ 45 s, and
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apo-AI (Luciani and Chimini, 1996; Hamon et al., 2000).
Genetic deficiency of ABC1 in humans causes Tangier disease, which is characterized by accumulation of phospholipids in the immune system with enlarged yellow tonsils and
hepatosplenomegaly (Bodzioch et al., 1999; Orsó et al., 2000).
ABC1 was also recently implicated in interleukin (IL)-1 processing and release (Hamon et al., 1997; Andrei et al., 1999),
because the release of IL-1 in response to extracellular ATP
is inhibited by glibenclamide and other ABC1 inhibitors
(Hamon et al., 1997), such as 4,4⬘-diisothiocyanostilbene2,2⬘-disulfonic acid (DIDS) and sulfobromophthalein (BSP).
IL-12 p40 is part of two heterodimeric cytokines that are
secreted mainly by activated antigen-presenting cells and
play a key role in determining the nature of the immune
response to exogenous or endogenous antigens. IL-12 is a
composite of IL-12 p40 and IL-12 p35 (Trinchieri, 1995),
whereas IL-12 p40 engages p19 to form IL-23 (Oppmann et
al., 2000). Both IL-12 and IL-23 enhance the proliferation,
cytotoxicity, and production of interferon (IFN)-␥ by T lymphocytes and natural killer cells (Trinchieri, 1995; Oppmann
et al., 2000), which is essential for the clearance of bacterial
infections (Trinchieri, 1995; Oppmann et al., 2000). Mice that
are genetically deficient in IL-12 p40 are highly susceptible
to infection with various intracellular pathogens (Mattner et
al., 1996). On the other hand, IL-12 p40 is an important
pathogenetic factor in autoimmune disease. This is demonstrated by the fact that although IL-12 p40-deficient mice are
resistant to collagen-induced arthritis (McIntyre et al., 1996),
transgenic overexpression of IL-12 p40 exacerbates the
course of this disease (Parks et al., 1998).
Because, as described above, IL-12 p40 plays a crucial role
in orchestrating the immune response, it is important to
investigate the cellular mechanisms that regulate the production of this cytokine. In this report, we demonstrate that
pharmacological inhibition of ABC proteins suppresses the
production of IL-12 p40.
ATP-Binding Cassette Transporters and Macrophage Activation
(JNK) (Promega, Madison, WI), anti-phospho-p38 MAP kinase (p38
MAPK; New England Biolabs, Beverly, MA), or an anti-inhibitory
factor ␬B (I␬B; Upstate Biotechnology, Lake Placid, NY) Ab and
subsequently incubated with a secondary horseradish peroxidaseconjugated donkey anti-rabbit Ab (Roche Molecular Biochemicals).
Bands were detected using ECL Western blotting detection reagent
(Amersham Biosciences, Inc.).
Measurement of Nitrite Concentration. Nitrite production, an
indicator of nitric oxide synthesis, was measured as previously described (Haskó et al., 1996) by adding 100 ␮l of Griess reagent (1%
sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric
acid) to 100-␮l samples of medium. The optical density at 550 nm was
measured using a Spectramax 250 microplate reader (Molecular Devices). The measurements of nitrite were performed using reagents free of
nitrite: no basal or background nitrite levels were detected.
Measurement of Mitochondrial Respiration. Mitochondrial
respiration, an indicator of cell viability, was assessed by the mitochondria-dependent reduction of MTT to formazan (Haskó et al.,
1996). Cells in 96-well plates were incubated with 0.5 mg/ml MTT for
60 min at 37°C. Culture medium was removed by aspiration, and the
cells were solubilized in 100 ␮l of DMSO. The extent of reduction of
MTT to formazan within cells was quantitated by measurement of
absorbance at 550 nm by using a Spectramax 250 microplate reader.
Detection of Surface I-Ad by Flow Cytometry. Peritoneal
macrophages were plated on 12-well plates and treated with glibenclamide (dissolved in 0.5% DMSO) in the presence or absence of
Fig. 1. Glibenclamide suppresses IL-12 p40 production in both J774 cells (A) and peritoneal macrophages (B). The cells were pretreated with various
concentrations of glibenclamide 30 min before LPS/IFN-␥ or LPS (in the peritoneal cells) stimulation, and IL-12 p40 concentrations were measured
from the supernatants collected 24 h after stimulation. Glibenclamide does not affect cell viability of the J774 cells as determined using the MTT assay
(C). Data are expressed as the mean ⫾ S.E.M. of six wells. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01.
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a final dwell at 72°C ⫻ 7 min; and ␤-actin, 5⬘-GAGACCTTCAACACCC-3⬘ (sense) and 5⬘-GTGGTGGTGAAGCTGTAGCC-3⬘ (antisense), an initial denaturation at 94°C ⫻ 5 min, 30 cycles of 94°C ⫻
30 s, 58°C ⫻ 45 s, 72°C ⫻ 45 s, and a final dwell at 72°C ⫻ 7 min.
With the exception of Kir6.2, in the absence of the reverse transcription
reaction, no bands were detected after the amplification. Because the
Kir6.2 primers amplified a product even without reverse transcription,
in the case of Kir6.2 RT-PCR, the RNA was treated with a DNA removal
kit from Ambion (Austin, TX). The expected PCR products were SUR1,
470 bp; SUR2A/B, 570/600 bp; Kir6.1, 477 bp; Kir6.2, 420 bp; ABC1, 422
bp; CFTR, 600 bp; and ␤-actin, 230 bp. PCR products were resolved on
a 1.5% agarose gel and stained with ethidium bromide.
Western Blot Analysis. Peritoneal macrophages in six-well
plates were pretreated with glibenclamide or vehicle, and 30 min
later the cells were stimulated with 10 ␮g/ml LPS for 15 min (Haskó
et al., 2000a,b). After washing with PBS, the cells were lysed by the
addition of radioimmunoprecipitation buffer. The lysates were transferred to Eppendorf tubes, centrifuged at 15,000g, and the supernatant was recovered. Protein concentrations were determined using a
Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). A sample (25– 40
␮g) was separated on 8 to 16% Tris-glycine gel (Novex, San Diego,
CA) and transferred to a nitrocellulose membrane. The blot was
conducted according to the ECL Western blotting protocol (Amersham Biosciences, Inc., Piscataway, NJ). The membranes were
probed with anti-phospho-mitogen-activated protein kinase (MAPK;
p42/p44, ERK1/2), anti-phospho-c-Jun N-terminal protein kinase
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Haskó et al.
Fig. 3. RT-PCR analysis of Kir6.1 and Kir6.2 mRNA expression in the
mouse heart (lane 1), NIT-1 cells (lane 2), the J774 macrophage cell line
(lane 3), the RAW 264 macrophage cell line (lane 4), and the mouse spleen
(lane 5). This figure is representative of three separate experiments.
IFN-␥ (100 U/ml; R & D Systems, Minneapolis, MN) for 48 h. Cells
were removed by scraping into 0.5 ml of Versene (Invitrogen) and
washed in PBS. After washing, the cells were resuspended in PBS
containing 10% mouse serum and Fc Block (rat anti-mouse CD16/
CD32; BD PharMingen, San Diego, CA) and then stained with a
fluorescein isothiocyanate-conjugated anti-I-Ad (BD PharMingen).
The cells were analyzed with a FACSCalibur flow cytometer (BD
Biosciences, San Jose, CA).
Statistical Evaluation. Values in the figures, tables, and text
are expressed as mean ⫾ S.E.M. of n observations. Statistical analysis of the data was performed by Student’s t test or one-way analysis
of variance followed by Dunnett’s test, as appropriate.
TABLE 1
Effect of DIDS, BSP, and DPC on IL-12 p40 production in J774
macrophages
DIDS and BSP suppress and DPC enhances IL-12 p40 production in J774 macrophages stimulated with 10 ␮g/ml LPS and 100 U/ml IFN-␥. Data are expressed as the
mean ⫾ S.E.M. of six wells.
Drug
IL-12
% of control
Control
10 ␮M DIDS
50 ␮M DIDS
100 ␮M DIDS
500 ␮M DIDS
10 ␮M BSP
50 ␮M BSP
100 ␮M BSP
500 ␮M BSP
300 ␮M DPC
1000 ␮M DPC
100 ⫾ 1.7
94 ⫾ 1.9*
47 ⫾ 5.2**
14.6 ⫾ 1**
0 ⫾ 0**
94 ⫾ 2*
57 ⫾ 2**
28 ⫾ 0.2**
5.8 ⫾ 0.1**
142 ⫾ 18*
179 ⫾ 24**
*p ⬍ 0.05; **p ⬍ 0.01.
Results
ABC Protein Inhibitors Suppress IL-12 p40 Production by both J774 Macrophages and ThioglycollateElicited Mouse Peritoneal Macrophages. To determine
whether ABC proteins are involved in the modulation of
IL-12 p40 production, we pretreated LPS/IFN-␥-stimulated
(data not shown). Similarly, the SUR inhibitors glipizide and
tolbutamide, as well as the SUR activators pinacidil, minoxidil, and diazoxide failed to alter the production of IL-12 p40
(data not shown). Finally, the selective CFTR inhibitor DPC
(Schultz et al., 1999) did not decrease, but rather enhanced
the production of IL-12 p40 (Table 1).
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Fig. 2. RT-PCR analysis of CFTR mRNA [top; mouse kidney (lane 1),
J774 cells (lane 2), peritoneal macrophages (lane 3), and mouse spleen
(lane 4)]. The bottom panel demonstrates mRNA expression of SUR1,
SUR2 A and B, ABC1, and ␤-actin in the mouse heart (lane 1), NIT-1 cells
(lane 2), the J774 macrophage cell line (lane 3), the RAW 264 macrophage
cell line (lane 4), and the mouse spleen (lane 5). This figure is representative of three separate experiments.
J774 macrophages with glibenclamide and measured IL-12
levels from the supernatants taken 24 h after the LPS/IFN-␥
challenge. The results of these experiments showed that glibenclamide inhibited the production of IL-12 p40 in a concentration-dependent manner (Fig. 1A). Glibenclamide did not
affect cell viability at the concentrations tested as determined with the MTT assay (Fig. 1C). We next investigated
whether the effect of glibenclamide could be reproduced using primary cells (peritoneal macrophages) instead of the
J774 macrophage cell line. Figure 1B shows that glibenclamide suppressed the production of IL-12 p40 also in peritoneal macrophages. Cell viability was not affected by glibenclamide in these cells (data not shown).
Having established that the inhibition of ABC proteins
suppresses cytokine production, we next examined whether
other ABC inhibitors can mimic the effect of glibenclamide.
First, we tested the two ABC1 inhibitors DIDS and BSP
(Becq et al., 1997; Hamon et al., 1997). Both of these inhibitors caused a concentration-dependent reduction of IL-12 p40
production (Table 1). On the other hand, 10 and 50 ␮M
verapamil, a P-glycoprotein inhibitor, was without effect
ATP-Binding Cassette Transporters and Macrophage Activation
107
Molecular Characterization of ABC Proteins in Macrophages. To further investigate which ABC proteins may
be involved in the regulation of IL-12 p40 production, we
conducted a series of RT-PCRs to determine which ABC
protein mRNAs are expressed in macrophages.
First, we determined whether mRNAs for SURs could be
found in macrophage cell lines. The heart was used as a
positive control for SUR2, because both splice variants (A
and B) have been shown to be expressed in the heart (Chutkow et al., 1996). The NIT-1 insulinoma cell line was the
positive control for SUR1, because SUR1 is the glibenclamide-binding protein found in pancreas ␤-cells (Babenko
et al., 1998). Figure 2 shows that neither SUR1 nor SUR2
transcripts were present in either the J774 or RAW 264 cells,
which rules out the possibility that glibenclamide suppresses
the production of IL-12 p40 by binding to an SUR. Because
glibenclamide has been shown to bind and inhibit Kir6 channels with a similar potency to its effect on IL-12 p40 production (Tucker and Ashcroft, 1998), we determined whether
any of the known Kir6 channels were expressed on macrophages. Figure 3 demonstrates that macrophages do not have
mRNAs for either Kir6.1 or Kir6.2.
Because human monocytes have been shown to transcribe
CFTR mRNA (Yoshimura et al., 1993), we determined,
whether this was the case in mouse macrophages. As shown
in Fig. 2, neither J774 nor RAW 264 macrophages expressed
CFTR mRNA. The kidney was used as a positive control,
because it has been shown to contain a large number of CFTR
transcripts (Marvao et al., 1998).
Finally, ABC1 mRNA was detectable in both macrophage
cell lines as well as in the spleen. In summary, these data
together with the results of the pharmacological studies suggest that ABC1, but not SURs, Kir6s, or CFTR is the target
of glibenclamide in macrophages.
Glibenclamide Prevents Intracellular Accumulation
of IL-12 p40 but not TNF-␣. Next, we asked the question
whether glibenclamide acts by decreasing the accumulation of
intracellular IL-12 p40 or it affects the release of IL-12 p40. The
results of this experiment showed that treatment of the cells
with LPS/IFN-␥ induced the appearance of both intracellular
and extracellular IL-12 p40, which were both suppressed to the
same extent by glibenclamide pretreatment (Fig. 4A). On the
other hand, glibenclamide did not influence the release of both
intracellular and extracellular TNF-␣ (Fig. 4B).
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Fig. 4. A, glibenclamide (glib) inhibits both the extracellular and intracellular accumulation of IL-12 p40. B, glibenclamide does not alter the
production of TNF-␣. C, glibenclamide decreases nitric oxide formation. J774 macrophages were pretreated with 100 ␮M glibenclamide and 30 min
later the cells were exposed to LPS/IFN-␥ for another 24 h. At the end of the incubation period, supernatants were collected and the adherent cells
were lysed for the determination of intracellular IL-12 p40 and TNF-␣. IL-12 p40 and TNF-␣ levels were determined by ELISA. Nitric oxide production
was measured from the cell supernatant by using the Griess method. Data are expressed as the mean ⫾ S.E.M. of eight wells. ⴱⴱ, p ⬍ 0.01.
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Haskó et al.
Fig. 5. Lack of effect of glibenclamide (glib) on LPS-induced degradation
of I␬B and the activation of p38, p42/44 MAPK, and JNK. Peritoneal
macrophages were pretreated with vehicle (cont) or 100 ␮M glibenclamide for 30 min followed by an LPS challenge for 15 min. The degradation of I␬B, and MAPK and JNK activation were determined using
Western blotting.
Discussion
The results of the present study demonstrate that the
inhibition of ABC proteins suppresses the production of IL-12
p40 but not TNF-␣ by activated macrophages. The suppression of macrophage activation by the inhibition of ABC proteins is not limited to LPS/IFN-␥-induced processes, because
IFN-␥-induced MHCII up-regulation is also attenuated by
the blockade of ABC protein function. Furthermore, previous
studies have shown that the blockade of ABC proteins also
impairs the production of IL-1␤ in macrophages induced with
ATP (Hamon et al., 1997; Andrei et al., 1999). It appears that
the mechanism by which ABC protein inhibition suppresses
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Glibenclamide Suppresses Nitric Oxide Production
by LPS-IFN-␥-Stimulated J774 Macrophages. To examine whether the glibenclamide suppression of macrophage
inflammatory mediator production was confined to IL-12
p40, we tested the effect of glibenclamide on nitric oxide
production. Figure 4C demonstrates that similar to IL-12
p40, the formation of nitric oxide was attenuated by glibenclamide.
Glibenclamide Fails to Alter LPS-Induced I␬B Degradation, MAPK, and JNK Activation. P42/44 MAPK,
p38 MAPK, and JNK are important intracellular components
of cellular responses to LPS and IFN-␥ (Firestein and Manning, 1999). Therefore, we tested whether the inhibitory effect of glibenclamide on IL-12 p40 production was due to an
interference with these pathways. Figure 5 shows that the
activation of these enzymes by LPS was not influenced by
pretreatment with glibenclamide.
The degradation of I␬B, the inhibitory part of the nuclear
factor ␬B-I␬B complex, is a central event in the transcriptional activation of a host of cytokine genes, including IL-12
p40 (Baeuerle and Henkel, 1994). Although LPS induced the
degradation of I␬B 15 min after stimulation, pretreatment
with glibenclamide failed to change I␬B degradation (Fig. 5).
Glibenclamide Suppresses IFN-␥-Induced Up-Regulation of Surface I-Ad Molecules in Peritoneal Macrophages. To further examine the effect of glibenclamide on
macrophage activation, we measured surface expression of
major histocompatibility complex (MHC) II molecules in response to IFN-␥. I-Ad expression was decreased, in a concentration-dependent manner, by treatment with glibenclamide
(Fig. 6).
Fig. 6. Glibenclamide (glib) suppresses IFN-␥-induced up-regulation of
MHCII molecules in peritoneal macrophages. Peritoneal macrophages
were plated on 12-well plates and treated with 100 ␮M glibenclamide in
the presence or absence of IFN-␥ for 48 h. MHCII expression was analyzed with a FACSCalibur flow cytometer. This figure is representative of
two separate experiments.
ATP-Binding Cassette Transporters and Macrophage Activation
cleotide binding domain characteristic of ABC proteins (Bankers-Fulbright et al., 1998).
Recent evidence indicates that glibenclamide also inhibits
both MDR and CFTR, as well as ABC1 (Schultz et al., 1996;
Ishida-Takahashi et al., 1998). The involvement of MDR in
the inhibitory effect of glibenclamide on IL-12 p40 production
is unlikely, because verapamil, a selective MDR inhibitor
(Hamon et al., 1997), failed to mimic the effect of glibenclamide on IL-12 p40 production. Similarly, verapamil did
not affect the release of IL-1␤ in ATP-stimulated macrophages (Hamon et al., 1997). CFTR does not appear to be
involved in the effect of glibenclamide, because we were unable to detect CFTR mRNA in the macrophages and the
CFTR blocker DPC did not decrease the production of IL-12
p40. Interestingly, CFTR-deficient epithelial cells failed to
express the chemokines regulated on activation, normal T
cell expressed and secreted, IL-8, and monocyte chemoattractant protein-1 in response to TNF-␣ stimulation (Schwiebert
et al., 1999). Furthermore, dysregulation of cytokine production was also observed in CFTR-deficient lymphocytes, where
the production of the anti-inflammatory cytokine IL-10 was
enhanced, and the release of the proinflammatory cytokine
IFN-␥ was decreased (Moss et al., 2000). These observations
suggest that CFTR regulates cytokine production in certain
cell types; however, this is not the case in mouse macrophages. On the other hand, it is possible that CFTR may
regulate cytokine production in human monocytes/macrophages, because these cells have been shown to express
CFTR (Yoshimura et al., 1993). The observations that ABC1
is expressed in macrophages, and that glibenclamide and the
other ABC1 blockers DIDS and BSP suppress ABC1 activity
in macrophages (Hamon et al., 1997) suggest that ABC1
could play a role in the inhibitory effect of these agents on
IL-12 p40 production. However, it is important to emphasize
that similar to glibenclamide, neither DIDS nor BSP is a
selective inhibitor of ABC1. For example, DIDS is a well
known purinoceptor antagonist (Ralevic and Burnstock,
1998), and BSP also inhibits the organic anion transporter
organic anion transporting polypeptide-1 (van Montfoort et
al., 1999). Clearly, further studies will be required to exactly
pinpoint the targets of the anti-inflammatory effect of glibenclamide, DIDS, and BSP.
In summary, this article demonstrates that macrophage
activation is inhibited by the blockade of ABC proteins by
glibenclamide as well as other ABC protein inhibitors. Because glibenclamide is widely used as an antidiabetic agent,
the effects of this drug on the immune system need to be
considered.
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